The invention relates to cellular detection. More specifically, the invention relates to the indirect detection of cells by the detection of ATP in a reaction mixture.
The ability to detect very low amounts of cells such as microbes is required in a variety of fields, both where sterilization is necessary and where the presence of microbes is expected as in foods and commercial water sources. By traditional methods, it is generally not possible to detect and quantify very small amounts of microbes directly from a sample. For example, a research scientist may want to know how many cells they have in a sample. A typical way of estimating this is to grow the cells in liquid culture and then to measure the optical density of the culture.
Reducing the amount of microbes to required levels is important in the making of consumer products, such as food and medical products. This is accomplished by autoclaving, irradiation, pasteurization, and filtration, among other techniques. A task of quality control is to ascertain the effectiveness of these techniques. Typically, such is the work of microbiologists, who attempt to grow cultures from swabs or samples of the product to be tested. On the other hand, commercial water sources are expected to contain low concentrations of microbes and microbiologists also grow cultures from these to determine their microbial counts in those sources.
In the food science and water treatment fields, liquid samples are typically analyzed using the Most Probable Number (MPN) method. The Most Probable Number method is basically a statistical analysis based on classical bacteriology culturing techniques, wherein in a range of dilutions of a liquid sample are inoculated into growth medium, and cell growth in the medium is detected. Standard MPN procedures often use a minimum of three dilutions and multiple culture tubes at each level of dilution. A typical worker would consult an MPN table to obtain the statistical multiplier to calculate the most probable number of bacterial cells in the original sample based on the growth distribution in the culture tubes.
Methods such as MPN are useful for calculating the number of living cells in a sample. The living cells in a consumer product are likely to grow during storage or after it is opened. The growth of these cells may adversely affect the consumer.
The time it takes to carry out an analysis by methods such as MPN that rely on the culturing of cells is a major drawback. The analyst must wait for the cultures to grow. Culture growth typically takes from overnight to several days.
All living organisms use adenosine triphosphate (ATP) as an energy source. In the prior art, cells are detected by assaying for the presence of ATP using the ATP-driven chemiluminescent (bioluminescent) luciferase/luciferin couple according to the following reaction.
luciferase
ATP+luciferin+O2xe2x86x92AMP+oxyluciferin+PPi+hv.
The light that is emitted from the reaction (hv) is conveniently measurable.
The amount of ATP present in a sample has been used to infer the amount of microbial organisms since the mid-1960s, according to U.S. Pat. No. 5,648,232, citing other work. Chittock et al., Analyt. Biochem., 255:120-126 (1998), discuss the detection of ATP on a food preparation surface as a marker for biological contamination. A firefly luciferase/luciferin system is used to detect the ATP that is present in a sample. In the art discussed in that publication, commercial systems have a detection limit of about 5 pg ATP. Chittock, et al. disclose a method to achieve that detection limit using a less sensitive luminometer and Michaelis kinetics to calculate the time at which the bioluminescence reaches one-half of its maximal value to calculate the initial ATP concentration. Chittock, et al. note that adenylate kinase activity can be driven by excess CTP, which is not a substrate of luciferase, and that adenylate kinase can convert AMP to ATP, citing Brovko et al., Anal. Biochem., 220:410-414 (1994).
U.S. Pat. No. 5,648,232 discloses a more sensitive way to detect microorganisms using a luciferase ATP assay. Rather than to detect only the ATP present at the time of measuring (linear relationship between photons produced and the amount of ATP), those workers detect the presence of adenylate kinase, an enzyme that catalyzes the conversion of ADP to ATP. By providing ADP, a single adenylate kinase molecule can give rise to an amplified amount of light by converting multiple ADP molecules to ATP and AMP. Those workers report that typically 400,000 ADP molecules are converted to ATP by a single adenylate kinase molecule in 10 minutes. The method disclosed in that patent for determining the presence and/or amount of microorganisms and/or their intracellular material present in a sample involves lysing the cells, providing adenosine diphosphate (ADP), luciferase/luciferin and preferably magnesium ions, and then analyzing for light produced from the luciferase/luciferin reaction.
An advantage to the method of U.S. Pat. No. 5,648,232 over mere analysis for the ATP that is present in a sample is that ATP can be present for a number of reasons, but the presence of adenylate kinase means that a living organism is likely present.
A drawback of that method is that solutions of ADP are unstable, resulting in formation of ATP. Thus, in a method of that patent, ADP is preferably kept in solid form until immediately prior to use. Contamination of the ADP, particularly with ATP, is highly undesirable for a cellular assay based on ATP detection. That patent advises purchasing high purity commercial ADP ( greater than 99.5% purity), then further purifying the ADP by column chromatography.
International Patent Application Publication No. WO/94/25619 discusses the art of the detection of biological material. Cyclic reactions for the measurement of low levels of NAD(H) or NADP(H) were discussed therein, citing EP-A-0060123, linearly amplifying the detection target, thus requiring a long time for sensitive measurements. WO/94/25619 cites GB-A-2055200 for disclosing a linear amplification of ATP using adenylate kinase to catalyse the reaction of AMP and ATP to form 2 ADP, which is then re-phosphorylated by pyruvate kinase to form ATP. In GB-A-2055200, the ATP is measured using bioluminescence and a luminometer.
The same cycle is discussed therein citing Chittock et al., Biochem. Soc. Trans., 19:160S (1991), wherein adenylate kinase forms two ADP from an AMP and an ATP. Pyruvate kinase then converts the two ADP to two ATP. Chittock et al. close the cycle by permitting luciferase to convert an ATP back to AMP, producing bioluminescence.
WO/94/25619 itself discloses a method of detecting ATP indirectly, by using ADP to convert glucose-6-phosphate to glucose with glucose kinase, then detecting the glucose product, the concentration of which increases exponentially when sufficient AMP is present with adenylate kinase to reform the consumed ADP. They add glucokinase and adenylate kinase and glucose-6-phosphate to determine ATP present.
It would be beneficial if a method were available for fast, highly sensitive detection of the presence of very low amounts of cells. The disclosure that follows provides such a method that also solves the problem posed by the instability of ADP by providing AMP and a high energy phosphate donor that are stable, as alternative precursors to ATP, and uses endogenous cellular enzymes to convert those precursors to ATP, as described below.
The invention contemplates a method for determining the presence and/or amount of cells in an aqueous sample composition. The method contemplates indirectly detecting cellular enzymes that convert AMP into ATP. The process includes providing a high energy phosphate donor, and preferably also providing AMP, and detecting the generated ATP. Such a method comprises the following steps. An aqueous sample solution to be assayed for the presence of cells is admixed with a high energy phosphate donor (other than ADP) to form a reaction mixture. The reaction mixture is maintained for a time period sufficient for enzymes endogenous to the cells to convert the AMP to ATP. The reaction mixture is assayed for the presence of ATP. An amount of ATP greater than that in a control indicates the presence of cells in the aqueous sample. Preferably, the conditions are favorable for phosphate transfer, such as an excess of phosphate donor relative to acceptor.
Preferably, the ATP is assayed using a Coleoptera-type luciferase enzyme, such as firefly luciferase, most preferably a thermostable luciferase. Preferably, the reaction mixture further comprises the luciferase. Preferably, the reaction mixture further comprises a high energy phosphate donor other than ADP, most preferably dCTP. It is also preferred to have magnesium ions in the reaction mixture.
In a preferred embodiment of the invention, the aqueous sample solution is a test sample wherein the cells have been treated by a physical method such as sonication or a series of freeze/thaw cycles or by admixture with a chemical agent or agents that lyse or otherwise disrupt the cellular membrane. Such a treatment serves to permeabilize or disrupt the membranes of the cells. Preferably the chemical agent or agents comprise an extractant, most preferably polymixin B sulfate and/or chlorhexidine.
In contemplated embodiments, the sample source is a solid or liquid. Contemplated sample sources are consumer products or materials used in the manufacture of a consumer product; examples include foods and beverages, cosmetics, medicinals and pharmaceuticals. It is further contemplated that the sample is a commercial or residential source of water.
In a further preferred embodiment, a sample source has been filtered to retain cells, thus forming a test sample. Most preferably, the cells on the filter, the test sample, are treated to form an aqueous sample composition. Thus a contemplated aqueous sample is a solid in contact with a liquid. In an alternative embodiment of such an aqueous sample composition, a solid sample source is analyzed directly by wetting the solid (admixing) with a solution containing a high energy phosphate donor, luciferase, and luciferin and detecting the light produced. Preferably, such a solid sample source is treated, most preferably by also admixing with an extractant.
The invention further contemplates a kit for the detection of cells that comprises purified high energy phosphate donor, preferably dCTP, purified luciferin and purified luciferase. Preferably, the luciferase is a thermostable luciferase such as Luc146-1H2. A contemplated kit further comprises purified AMP, and optionally, magnesium ions. It is further contemplated that the kit comprises an extractant, preferably comprising polymixin B sulfate and/or chlorhexidine.
The present invention has many benefits and advantages, several of which are listed below.
One benefit of the invention is that its use provides results more rapidly than classical culturing methods.
An advantage of the invention is that it is very sensitive, able to detect as few as 4 cells in an aqueous sample.
Another benefit of the invention is that that the cellular detection is possible despite a large background of ATP in the sample source.
Yet another advantage of the invention is the enhanced stability of the reagents for cellular detection.
Still further benefits and advantages of the invention will become apparent from the specification and claims that follow.
To facilitate understanding of the invention, a number of terms are defined below.
The term xe2x80x9cisolatedxe2x80x9d when used in relation to an organic molecule such as luciferase, luciferin, AMP, or dCTP, refers to a molecule that is identified and separated from at least one contaminant with which it is ordinarily present. Thus, an isolated molecule is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated molecules are found in the state they exist in nature.
As used herein, the term xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cto purifyxe2x80x9d means the result of any process that removes some contaminants from the component of interest. The percent of a purified component is thereby increased in the sample.
The term xe2x80x9csample source,xe2x80x9d as used herein, is used in its broadest sense. A xe2x80x9csample sourcexe2x80x9d is suspected of containing a cell or is an intermediate to the formation of the xe2x80x9caqueous samplexe2x80x9d that is analyzed. The phrase xe2x80x9caqueous samplexe2x80x9d contemplates a liquid solution, an emulsion or a suspension, and a solid in contact with a liquid. The location of the aqueous sample is not limited to a traditional reaction vessel. For example the solid sample source may be a solid or porous material supporting a liquid film. A xe2x80x9ctest samplexe2x80x9d contemplates a sample that is derived from a sample source
The term xe2x80x9cdetection,xe2x80x9d as used herein, refers to quantitatively or qualitatively determining the presence or absence of a component within a sample.
The present invention contemplates a process for the indirect detection of the presence of cells in an aqueous sample by detecting light produced from ATP that is generated in the reaction from AMP and a high energy phosphate donor using endogenous cellular enzymes admixed with exogenously supplied AMP a high energy phosphate donor.
A process of the invention differs from known cellular detection methods in that it relies upon cellular endogenous enzymes to use the provided high energy phosphate donor to produce a light signal. In preferred embodiments, the more stable AMP is provided, as opposed to the relatively unstable ATP precursor, ADP, as provided in a method of U.S. Pat. No. 5,648,232.
A method of the invention is able to detect the presence of cells that are viable or only recently degraded. Because a process of the invention uses endogenous cellular enzymes, loss (or absence) of the activity of those enzymes results in the lack of a signal. Cell death, for example as a result of most sterilization processes tends to result in enzyme inactivity. Cells tend to lyse upon death, and enzymes once contained in intact cells will denature or are degraded.
The types of cells that can be detected by a process of the invention include: prokaryotes such as bacteria, eukaryotic cells, archael cells, fungi, plant and animal cells. A method of the invention is particularly useful for detecting bacterial contamination. As noted before, all living things use ATP as an energy source and therefore have enzymes capable of catalyzing the conversion of AMP and a high energy phosphate donor to ATP as required by the process of the invention.
The invention contemplates that samples containing a very large number of cells are diluted, preferably serially, to come within accurate ranges of detection when the amount of cells present is sought. A contemplated method is very sensitive, as shown in the Examples herein.
The process for detecting the presence of cells taught herein is not useful for the determination of the kinds of cells (speciation) with the exception of the distinction between eukaryotic and prokaryotic cells as discussed herein with regard to the use of certain extractants. For a determination of the types of cells present, useful processes are disclosed in the parent, copending applications U.S. Ser. No. 09/252,436, filed on Feb. 18, 1999, which is a continuation-in-part of U.S. Ser. No. 09/042,287, filed Mar. 13, 1998.
The detection of ATP using a contemplated process can be quantitative. The addition of known quantities of ATP to samples prepared according to the invention result in standard curves that are useful for the determination of ATP concentrations in the samples. The linear response range exists for several powers of ten. The addition of ATP directly to a sample results in an additive effect on the light signal produced.
Adenosine exists in a cell in any of its three interconvertible forms: AMP, ADP and ATP. The combined total of the three is the xe2x80x9cadenosine poolxe2x80x9d of a cell. The relative amounts of the three forms depends upon the energy state of the cell, for example dormant cells have less of the ATP form. It was known in the art to detect cells by measuring the ATP form that was exogenous in a cell. Thus, it was more difficult to detect dormant cells than active cells. The present invention solves that problem by providing a high energy phosphate donor and permitting endogenous enzyme activity to convert the entire adenosine pool to the ATP form.
Some workers in the art suggested enhancing a cellular ATP detection signal by adding exogenous ADP and permitting endogenous adenylate kinase and phosphate donors to convert the added ADP to additional ATP. The present invention contemplates the provision of AMP in addition to the high energy phosphate donor to enhance ATP-generated light output.
The cellular adenosine pool provides insight into the cellular enzyme concentration, and thus the number of cells present. As shown in the Examples hereinafter, the light signal produced is proportional to the number of cells in a sample. Thus, a process of the present invention is useful for the determination of the presence and/or amount of cells in a sample.
The present invention provides methods for determining the presence and/or amount of cells and cellular material present in the sample. In some embodiments of this invention, cells are treated to make the added phosphate donor (D-P) accessible to endogenous enzymes. In other embodiments, high energy phosphate donor molecules and AMP molecules are then added to the treated cells. It is believed that the primary pathway resulting in ATP formation proceeds through the formation of ADP molecules. ADP molecules produced by the enzymatic transfer of a phosphate group from the high energy donor to AMP according to the following reaction:
Dxe2x88x92P+AMPxe2x86x92D+ADP
The reaction is catalyzed by endogenous enzymes present in the reaction mixture. It is further believed that a primary pathway for ATP formation includes the enzymatic transfer of a phosphate from the donor molecules to adenosine 5xe2x80x2-diphosphate molecules according to the following reaction:
Dxe2x88x92P+ADPxe2x86x92D+ATP
The ADP phosphorylation reaction is also catalyzed by endogenous enzymes present in the reaction mixture. Thus, the endogenous cellular components provide various enzymatic activities such as nucleotide monophosphate kinase (NMPK) activity and nucleotide diphosphate kinase (NDPK) activity, whose combined effect results in the production of ATP. In particularly preferred embodiments, the ATP is then detected by either a luciferase detection system or NADH detection system. The AMP are endogenous or can be exogenously supplied as discussed hereinabove.
A process of the invention utilizes an aqueous sample composition. The sample composition can be made from a variety of sample sources, including test samples. Samples can be obtained from liquids, such as inter alia beverages, drinking water, liquid waste and runoff. Test samples can also be obtained from gases for example, by adhesion of the cells or microorganisms to a solid surface and then extracting them or the cell contents into a solution, or by bubbling the gas through a liquid, for example an extractant. U.S. Pat. No. 5,773,710 provides an apparatus for such gas sampling. Test samples can also be taken from solids for example, by swabbing a surface (e.g. a manufacturing surface) and swirling the swab in a liquid for analysis, or by suspension of a solid sample (e.g. a food sample) in a liquid.
The invention contemplates that the reactions typically occur in solution although some bacterial sample sources are a solid, for example by swabbing a surface or forcing air through a filter. It is contemplated that for such a solid sample source, an aqueous sample is made by adding the filter to the extractant to permit the high energy phosphate donor access to the enzymatic activities needed to perform the necessary phosphate transfers. Alternatively, the cells may be treated directly on a solid surface providing for detection from the surface.
A process of the invention is useful for quality control and monitoring. The process is useful in the food processing and manufacturing, waste treatment, and medical fields. Gas sampling is useful in manufacturing under sterile conditions, military biological warfare monitoring, and hospitals and treatment wards. Solid sampling is useful in manufacturing and medical fields.
Treatment to lyse or disrupt a cell is not essential in a process of the invention. However, it is preferred to at least permeabilize the cell, thereby leading to better access to endogenous enzymes. Such treatment also enhances the reproducibility of results from a process of the invention. Thus, for optimum detection of cells, it is preferred to disrupt or otherwise break down the cells so that intracellular enzymes for converting AMP to ATP are released or otherwise exposed to the provided reagents.
Methods for permeabilization, lysis or disruption of cells are well known in the art. A wide variety of equipment is available for mechanical disruption, including sonicators (ultrasonic generators) and French presses. Cells can be disrupted by osmotic shock, by treatments such as a series of freeze-thaw cycles, or a rapid alteration of the ionic strength of the environment, or by the use of agents that directly disrupt cell membranes such as enzymes like lysozyme or chemical agents such as detergent or surfactants and antibacterials such as polymixin B and chlorhexidine.
Such chemical reagents are commercially available and commonly referred to as xe2x80x9cextractantsxe2x80x9d. Typical extractants include general cationic detergents such as CTAB (cetyl trimethyl ammonium bromide), anionic detergents such as sodium dodecyl sulfate and nonionic surfactants such as poloxyethylene alkyl phenyl ethers (e.g. Tritons(copyright), from Sigma, St. Louis, Mo.), nonoxynols, or other materials such as polymixin B sulfate or chlorhexidine, and proprietary formulae such as Extractant(trademark) (F352A, Promega Corp., Madison, Wis.) and Celsis-Lumac (1290142, Celsis, Evanston, Ill.).
A typical concentration for such detergents for use in disrupting cells ranges from about 0.01% to 10.0% in an aqueous solution. Cationic detergents are known to release the contents of eukaryotic cells as well as all other kinds of cells. In contrast, the use of a non-ionic detergent is effective for releasing materials from eukaryotic cells without disturbing other kinds of cells. Thus, one can distinguish between bacterial cells versus eukaryotic cells.
The Examples hereinafter show that some extractants or combinations thereof affect more than others the light signal produced from the luciferase/luciferin couple, and thus the sensitivity. Such effects can be lessened using other chemical additives. For very sensitive cellular detection, a preferred cell treatment solution combines about 0.05 to about 2 mg/mL of polymixin B sulfate with about 0.01 to about 10 percent chlorhexidine, preferably about 0.5 to about 1.5 mg/mL polymixin B sulfate with about 0.2 to about 6 percent chlorhexidine, and most preferably about 1 mg/mL polymixin B sulfate and about 0.05 percent chlorhexidine. Preferably, the chlorhexidine is added just prior to use.
Some variation in the sensitivity of detection of the same type of cells between stationary phase cultures and log phase cultures is reported below in Example 22. It is noted that the presence of the cells is detected using a method of the invention in either situation.
Filtration is desirable in some cases as a step in testing a liquid or gaseous sample by a process of the invention. A filtration step can serve two purposes i) collection of cells, and ii) isolation of cells. Filtration is preferred when a sample is taken from a large volume of a dilute gas or liquid.
Filtration serves to minimize sampling error from dilute sources. One skilled in the art understands that with dilute sources, xe2x80x9csampling errorxe2x80x9d is not really a measurement error, but rather it is a reality of the sample. When a liter of solution contains only 5 cells, there are many one milliliter samples of that liter that cannot contain a single cell. Thus, the larger the sample, the smaller the error. Filtering a large volume permits a concentration of the cells from within that large volume into a known small volume.
Because of the sensitive detection method used herein, such concentration is not always required to achieve a signal. As is evident from the Examples hereinafter, dilution of the resuspension from the filter is often desired before luminescence detection.
Filtering also serves to isolate the cells from their source. This isolation is often desirable for sensitive, reproducible assays. As the Examples hereinafter demonstrate, there are some components of a sample source that can inhibit light generation from the luciferase/luciferin reaction. A filtration step can also serve to separate the sought after cells from the offending component of the sample source.
Similarly, a filtration step can be used to transfer the cells to be assayed to a solution that provides a lower background. For example, a source with a large amount of ATP outside of the cells can be filtered to isolate the contaminating bacteria, and then the bacteria can be washed to further remove adhering ATP from the source. For example, fruit juices contain very high amounts of xe2x80x9cbackgroundxe2x80x9d ATP.
Filtration materials and methods for the removal or isolation of cells are well known to those in the art. Materials have advanced significantly since the 1970s from the standpoint of the ability to retain microorganisms effectively while passing a large volume relatively rapidly. Advanced manufacturing techniques make available highly homogenous separation materials. A large array of filters are available with a variety of size cut-offs and membrane hydrophobicities and charges. Thus, one can select for the retention of certain types of microorganisms over others.
One skilled in the art will appreciate that many of the materials and methods for the isolation of cells can be substituted for a filtration step in preparing a sample for analysis by a contemplated process. Such methods include immunological concentration of cells, centrifugation, as well as buffer exchange or washing to remove background signal contributors. Dialysis methods serve equally well for removal of background signal contributors, and to a certain extent for concentration.
For many applications of a process of the invention, standard 0.2 micron cut-off membranes (e.g. 0.22 micron Millipore(copyright) Express Membrane, Millipore Corp., Bedford, Mass. or those available from Fisher Scientific, Pittsburgh, Pa.) can be used.
As mentioned above, washing of the isolated cells, preferably with a non-disrupting low or no-background solution is an optional step to decrease background signal. Exemplary washes include buffers for the extractant omitting the extractant, or a Tris-buffered saline solution. Typically, the wash buffer is not such as to cause osmotic shock (lysis) or otherwise disturb the membrane integrity of the cells or microorganisms.
The luciferase/luciferin reaction is well known in the art, and there are commercial sources for the necessary reagents as well as protocols for their use. For example, several luciferase/luciferin reagents along with luciferase are available from Promega Corp., Madison, Wis. Commercially available luciferases include firefly luciferase (Photinus pyralis, xe2x80x9cPpy luciferasexe2x80x9d). Purified beetle luciferin is also commercially available from Promega.
Other luciferases useful in a process of the invention include thermostable luciferases, such as the thermostable Luc90-1B5 luciferase, and other mutants, Luc133-1B2 luciferase and Luc146-1H2 luciferase. Thermostable luciferases are preferred in a process of the invention because they are resistant to the destabilizing effect of the materials used to permeabilize the cells, i.e. chemicals such as chlorhexidine. Further, contaminating adenylate kinase activity can often be easily reduced from a luciferase preparation by heat denaturation of the adenylate kinase (e.g. from the non-thermostable cell in which a recombinant luciferase is produced). The thermostable Luc146-1H2 luciferase is disclosed in copending U.S. Patent Application, xe2x80x9cThermostable Luciferases and Methods of Productionxe2x80x9d, filed Sep. 15, 1999, is a continuation-in-part of U.S. application Ser. No. 09/156,946 filed in December 1998, the disclosures of both of which are incorporated herein by reference. Embodiments of the invention using thermostable luciferases are preferred.
It has been found that the stability of the luciferase enzyme is enhanced by the addition of a stabilizer, such as bovine serum albumin (BSA) or gelatin. BSA or gelatin may also have a stabilizing effect on the cellular enzymes that are responsible for the phosphate transfer. Due to the tendency of commercially available BSA to contain adenylate kinase, ATP and other impurities, it is preferable to use gelatin (0.1 to 2 percent by weight) in a contemplated process.
The luciferase/luciferin reaction is well-known in the art for the detection of ATP. The reaction conditions are also well known. Several exemplary reaction conditions are in the Examples below and in the parent applications listed above, incorporated herein by reference.
In a contemplated embodiments, ATP detection reagent referred to as L/L reagent (Promega, FF2021) is utilized. Most preferably, the luciferase/luciferin reagent comprises a thermostable luciferase in a compatible buffer containing luciferin, such as the solution that is used in Examples 20-23, below.
The amount of luciferase used in a 100 xcexcL reaction is 1-100 xcexcg, preferably, about 1 to 10 xcexcg.
The pH value of the reaction should be between about pH 4.5 and pH 9. The currently available luciferase enzymes are active in that pH range. Low activity was noted at pH 4.5 in Example 20. Preferably, the pH value is about pH 7 to about pH 8, most preferably, about pH 7.2. The temperature range is preferably about zero degrees C to about 80xc2x0 C. In embodiments of the invention using non-thermostable luciferases, the reaction temperature is preferably about 15xc2x0 C. to about 35xc2x0 C., most preferably about 22xc2x0 C. In preferred embodiments using the thermostable luciferases, a pretreatment temperature to remove contaminating enzyme activity is preferably about 50xc2x0 C. to about 80xc2x0 C., most preferably about 65xc2x0 C., and the reaction temperature for the thermostable enzymes is as with the non-thermostable luciferase enzymes. A heat pretreatment step is preferably one to ten minutes long, however if contaminating enzyme activity such as adenylate kinase activity is still higher than desired at that time, a repeat or longer time is useful.
When a sample is taken from a solution that does not interfere with the luciferase reaction, there is no need to wash the cells prior to analysis. Phenol red can interfere with the luciferase reaction. Extractant(trademark) (Promega F3532A) inhibits the reaction slightly, but that affect is ameliorated by the presence of Triton(copyright) N-101 (nonoxynol-10). Chlorhexidine also negatively affects the light production, and that negative affect is also ameliorated by Triton(copyright) N-101. Coenzyme A was found to slightly negatively affect the reaction results.
Although it is recognized that proper controls can account for impurities in the reagents and buffers, it is preferable to use reagents with as high a purity as possible for all reagents. The presence of ATP, or particularly, enzymes such as adenylate kinase (both common in luciferase preparations) can skew the results and cause a high background signal.
Preferably, magnesium ions are provided in a process or kit according to the invention, most preferably as MgCl2, Mg(OCOCH3)2, or MgSO4. It is recognized by those skilled in the art that other forms of magnesium ions serve equally well, and further that other ions can serve as substitutes for magnesium ions (e.g. Mn2+ or Ca2+). It is also noted that magnesium ions tend to be ubiquitous to the extent that the addition of exogenous magnesium ions is not necessary to practice the invention. However, the provision of magnesium ions is preferred for maximal reproducibility and sensitivity.
As is evident from the luciferase/luciferin reaction above, oxygen is a reactant of the reaction. Therefore, the reaction should not be conducted under anaerobic conditions. However, it is not generally necessary in practicing the invention to provide oxygen over an above that present in the air. Reactions can take place in closed vessels, provided there is sufficient oxygen in the reaction solution.
As shown in the Examples hereinafter, the luciferase reactions can be conveniently conducted in a plate with multiple reaction wells. The light readings can be conducted therein with the proper equipment.
The preparation of the assay preferably involves the preparation and running of appropriate controls, preferably including a sample that does not contain permeabilizing agents. Another desirable control is a heat treated sample that is devoid of background adenylate kinase activity.
In a contemplated process of the present invention, enhanced sensitivity of a cellular detection process of the invention is a result of the addition of exogenous adenosine monophosphate (AMP) and a high energy phosphate donor. The exogenous AMP is converted by endogenous cellular enzymes, in the presence of a high energy phosphate donor, into ATP, which is detected using the luciferase/luciferin reaction, thereby showing the presence of cells or microorganisms.
In an embodiment of the invention, a high energy phosphate donor for AMP is provided that is not a preferred substrate for luciferin. Moyer and Henderson, Anal. Biochem., 131:187-189 (1983), note that although ATP is the preferred substrate for firefly luciferase, other nucleotides dATP, XTP, UTP, GTP, TTP, dUTP, CTP, dGTP, ITP, dITP, dCTP can act as substrates, but are less than {fraction (1/50)} as active as ATP. Moyer and Henderson concluded that these other nucleotides can contribute to the background but are negligible with respect to their effect on ATP detection. Thus, any of those nucleotides can be used as high energy phosphate donors for the conversion of AMP to ATP.
The present invention provides methods utilizing different substrates for detecting the presence of cells or microorganisms in a liquid sample (preferably a lysate) suspected of containing cellular material or microorganisms. The system possibly takes advantage of a coupled reaction catalyzed by endogenous NMPK activity and NDPK activity according to the following reaction scheme:
AMP+Dxe2x88x92Pxe2x86x92D+ADP and
ADP+Dxe2x88x92Pxe2x86x92ATP+D
wherein Dxe2x88x92P is a high energy phosphate donor added to the cell lysate and AMP is adenosine monophosphate added to the cell lysate sample. In this reaction, ADP molecules are produced by the enzymatic transfer of a phosphate group from the high energy phosphate donor molecules (Dxe2x88x92P) to the added AMP molecules. Then, ATP is produced by the enzymatic transfer of phosphate from Dxe2x88x92P molecules to the ADP molecules according to the general reaction described above that is catalyzed by endogenous enzymes present in the sample.
The conversion of AMP to ATP by the endogenous enzymes, when present, can take place very rapidly, so one can detect the cell very rapidly. However, when detecting very low numbers of cells, it is preferable to obtain light reading data points after at least 10 minutes of incubation of the AMP with the endogenous enzymes. There is no negative effect resulting from incubation of the exogenously supplied AMP with the endogenous enzymes in the presence of the luciferase and luciferin.
Co-optimization of the concentrations of AMP and the high energy phosphate donors added to the samples is helpful to optimize light output from these reactions. The concentration range for the added AMP in the reaction mixture is about 0.001 mM to about 100 mM AMP. For the high energy phosphate donor, the concentration range is about 0.01 mM to about 100 mM. In preferred embodiments, the high energy phosphate donor is dCTP. Preferably, the high energy phosphate donor is provided in equal amounts or more preferably in excess relative to the concentration of adenosine phosphate acceptor. In a preferred embodiment, a reaction mixture has about 0.01 mM to about 50 mM added AMP and about 0.1 mM to about 50 mM added dCTP. In particularly preferred embodiments, addition brings the concentrations in the reaction mixture to about 0.1 mM AMP and about 1 mM dCTP.
After addition of nucleotides to the sample, the samples are incubated from 0 minutes to 3 hours, preferably incubated for about 10 to 60 minutes. The light output from the samples is determined preferably by luminescence measurement.
Other preferred buffers and reactions components can be found in the Examples.
The present invention provides important advantages over previously described cell detection systems. AMP is much more stable than ADP, and results obtained using the present invention are more reproducible than previously used methods.
As discussed above, a separate luciferase/luciferin reagent can be added before or significantly after addition of a high energy phosphate donor to the sample. Optionally, the luciferase/luciferin stock can be formulated with the high energy phosphate donor present.
The present invention provides test kits for determining the presence of cells in a test sample. In preferred embodiments, the test kits comprise the essential reagents required for the method. In some preferred embodiments, these reagents include, but are not limited to, a high energy phosphate donor (other than ADP) which is not efficiently used by luciferase (preferably dCTP) and AMP together with luciferase and luciferin. In alternative preferred embodiments, the kit includes all these reagents with luciferase and luciferin being provided in the same solution.
In other preferred embodiments, the reagents are free of contaminating components, including, but not limited to, adenylate kinase and ATP (i.e., contaminants that can cause a false positive result).
In still other preferred embodiments, a cell treatment (permeabilization or lysis) cocktail can be provided for efficiently releasing the contents of the target cells for each of the assays intended. In some embodiments for detecting prokaryotic microorganisms, only a cationic detergent is needed. In yet other embodiments for fungal spore, yeast, or eukaryotic cells assays, a nonionic detergent reagent is included. In preferred embodiments, reagents are provided in vessels and are of a strength suitable for direct use or use after dilution. In particularly preferred embodiments, a buffer solution for diluting the cell samples can also be provided. A contemplated kit optionally includes printed instructions.